AU750322B2 - Methods and compositions for reducing ischemic injury of the heart by administering adenosine receptor agonists and antagonists - Google Patents

Description

WO 98/50047 PCT/US98/09031 METHODS AND COMPOSITIONS FOR REDUCING ISCHEMIC INJURY OF THE HEART BY ADMINISTERING ADENOSINE RECEPTOR AGONISTS AND ANTAGONISTS Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health, Grant Number HL48225.

FIELD OF THE INVENTION The present invention relates to therapeutic methods for protecting the heart from ischemic injury.

More specifically, the methods of the invention involve administering agonists and antagonists or binary conjugates thereof which selectively activate or inhibit adenosine receptors simultaneously thereby enhancing the protective effects of preconditioning and rendering the myocardium more resistant to ischemia.

BACKGROUND OF THE INVENTION Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.

Adenosine is released in large amounts during myocardial ischemia and can mediate potentially important protective functions in the cardiovascular system (1,4,5,7,9,14, 17,18,19,25). Previous studies have shown that adenosine receptor agonists can precondition the heart when given before the onset of ischemia (4,5,9,14,17,18) and can cause reduction in WO 98/50047 PCT/US98/09031 infarct size or improvement in left ventricular function when given during reperfusion (1,19) or during both low-flow ischemia and reperfusion in isolated perfused heart (6,21,22). While activation of adenosine Al and A3 receptors has been shown to mimic the cardioprotective effect of preconditioning (3,10,23,24), their roles in mediating the protective effect of adenosine administered during ischemia have not yet been fully elucidated. Further, the cardioprotective effect of exogenous adenosine infused during ischemia in the intact heart may be exerted at the level of coronary vasculature, circulating neutrophils, or cardiac myocytes.

Our previous studies have characterized a cardiac myocyte model of injury, which is induced by exposure of myocytes to prolonged hypoxia in glucose-free media (16,23). Use of this model has facilitated the identification of compounds that enhance the protective effects of preconditioning and also increase myocardial resistance to ischemia.

SUMMARY OF THE INVENTION The present invention provides methods for preventing or reducing ischemic damage of the heart. In conducting research leading up to this invention, it was discovered that simultaneous activation of A3 and Al receptors enhances the protective effects of preconditioning and increases myocardial resistance to ischemia. The concept underlying the present invention is the use of specific agonists which simultaneously activate these two adenosine receptors. Concomitant activation of the two receptors is believed to produce a synergistic effect enhancing the cardioprotective effects of preconditioning and increasing myocardial resistance to ischemia.

According to a preferred embodiment, the invention involves administration of specific A1/A3 agonists, such WO 98/50047 PCT/US98/09031 as N 6 (2-trifluoromethyl) (carbamoyl) adenosineor N 6 -(3-iodophenyl)(carbamoyl) during ischemic attacks, or at risk for ischemic damage. The agonists of the invention may be delivered prior to a surgical procedure. They may also be administered to a patient to prevent or reduce the severity of ischemic damage during surgery.

Additionally, the A3/A1 agonists may be administered following surgical procedures to reduce the risk of post-surgical ischemic complications. Finally, the A3/A1 agonists may be administered to patients with angina or to patients during a myocardial infarction. The angina may be chronic and stable, unstable, or post-myocardial infarction angina.

In yet another embodiment of the invention, a series of water-soluble MRS compounds are contemplated to be within the scope of the present invention. These compounds selectively activate the A3 receptor. Because the compounds of the invention do not cross the blood-brain barrier, the deleterious effects associated with A3 receptor activation in the brain are avoided.

The MRS compounds will be used in conjunction with the Al agonists of the invention to prevent or reduce ischemic damage to the heart.

Another preferred embodiment of the invention comprises novel binary conjugates which bind two adenosine receptors simultaneously. Exemplary binary conjugates of the invention contain moieties that act as agonists at both of the Al and A3 adenosine receptors, such as MRS 1543. A second exemplary conjugate, MRS 1528, acts simultaneously as an agonist at the A3 receptor and as an antagonist at the A2a receptor.

Methods are disclosed herein for the administration of these binary conjugates to protect the heart against ischemic damage.

Methods of simultaneous administration of the A3 and Al agonists or the binary A3 agonist/A2a antagonist WO 98/50047 PCT/US98/09031 or the binary A3 agonist/Al agonist of the invention include direct perfusion of the organ during surgery and intravenous administration. Additionally, the agonists and antagonists of the invention may be administered to patients in tablet form in an amount effective to prevent or reduce ischemic damage to the heart.

In yet a further aspect of the invention, recombinant myocytes are provided which may be used to advantage in assessing the activity of agents that may possess cardioprotective activity. Cardiac myocytes may be transfected with any of the adenosine receptor encoding cDNAs and used to screen for novel therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B are graphs showing the effects of adenosine A3 receptor antagonists on the 2-chloro-N 6 -cyclopentyl-adenosine (CCPA) and 2-chloro-N 6 -(3-iodobenzyl)adenosine- (Cl-IB-MECA)-induced cardioprotective effect. Cultured ventricular myocytes were prepared and the extent of hypoxia-induced myocyte injury determined as described hereinbelow. The A3 antagonist, 3-ethyl- 5-benzyl-2-methyl-6-phenyl -4-phenylethynyl- 1,4-(1)-dihydropyridine-3,5dicarboxylate (MRS 1191) was present at the indicated concentrations individually, with CCPA (10 nM), or with Cl-IB-MECA (10 nM) during the ninety-minute hypoxia.

The percentage of myocytes killed, Figure 1A and the amount of CK released, Figure, 1B, were determined following the prolonged hypoxia. Data represent the mean ±SE of three experiments.

Figure 2 is a graph showing effects of adenosine Al agonists, 2-chloro-N 6 -cyclopentyladenosine

(CCPA),

N

6 -cyclohexyladenosine (CHA), and adenosine amine congener (ADAC) on cardiac myocyte injury in the WO 98/50047 PCT/US98/09031 presence or absence of excess 8-cyclopentyl-l,3dipropylxanthine (DPCPX), an Al antagonist. (open squares, CCPA; open triangles, CHA, open circles, ADAC; filled squares CCPA DPCPX; filled triangles, CHA DPCPX; filled circles, ADAC DPCPX) Figures 3A-3F are graphs illustrating the cardioprotective effects of the MRS compounds of the invention. Figure 3A shows the amount of creatine kinase released as a function of increasing concentrations of MRS 584 in the presence (diamonds) or absence (open squares) of the Al receptor antagonist DPCPX. Figure 3B shows the amount of creatine kinase released as a function of increasing concentrations of MRS 537 in the presence (diamonds) or absence (open squares) of the Al receptor antagonist DPCPX. Figure 3C shows the amount of creatine kinase released as a function of increasing concentrations of MRS 479 in the presence (diamonds) or absence (open squares) of the Al receptor antagonist DPCPX. Figure 3D shows the amount of creatine kinase released as a function of increasing concentrations of MRS 1340 in the presence (diamonds) or absence (open squares) of the Al receptor antagonist DPCPX. Figure 3E shows that both MRS584 and MRS537 can exert the cardioprotective effect of preconditioning via the human adenosine A3 receptor. Data represent means SE of three experiments. Figure 3F shows that Cl-IB- MECA reduces the percentage of myocytes killed during ischemia.

Figures 4A, and 4B are graphs showing the reduction in cardiac myocyte cell death following simultaneous exposure to Al and A3 agonists on cardiac cells. Figure 4A shows that the preconditioning effect is synergistically enhanced when adenosine Al and A3 receptors are activated simultaneously, as opposed to activation of a single receptor. The percentage of WO 98/50047 PCT/US98/09031 myocytes killed is also significantly reduced in the simultaneous presence of Al and A3 agonists during prolonged ischemia. See Figure 4B. Data represent means SE of four experiments.

Figures 5A- 5D are graphs showing the cardioprotective effects mediated by MRS 646 and MRS 1364 which activate the Al and A3 receptors simultaneously. Figure 5A shows that MRS646 and MRS1364 can pharmacologically precondition the cardiac myocyte.

The extent of myocyte injury was quantitated as percentage of myocytes killed. The data show that when either MRS646 or MRS1364 was present during the injury-producing ischemia, both compounds protect against injury. See Figure 5B. Figure 5C shows that MRS 1364 was able to pharmacologically precondition the cardiac myocytes transfected with the human adenosine A3 receptor. Data represent the means S.E. of three experiments. Figure 5D shows that MRS 646 was able to pharmacologically precondition the cardiac myocytes transfected with the human adenosine A3 receptor. Data represent the means S.E. of three experiments.

Figures 6A, 6B and 6C are graphs that show the cardioprotective effects of MRS1543, a binary A1/A3 agonist. Figure 6A shows that the protective effect of MRS1543 was only partially attenuated by DPCPX or by MRS1191. Ventricular myocytes were exposed to the indicated concentrations of MRS1543 in the presence or the absence of excess DPCPX (1 MM) or excess MRS1191 (1 AM). The ventricular myocytes were also exposed to the indicated concentrations of MRS1543 in the presence or the absence of excess DPCPX (1 MM) plus excess MRS1191 (1 MM). See Figure 6B. The combined presence of both DPCPX and MRS1191 completely abolished the protective effect of MRS1543. The protective effect of MRS1543 in chick cells transfected with the human adenosine A3 WO 98/50047 PCT/US98/09031 receptor is shown in Figure 6C. Data represent means SE of three experiments.

Figures 7A and 7B are graphs showing the cardioprotective effects of simultaneous administration of an A3/A1 agonist, MRS 580, and an A 2 a antagonist on myocyte survival. Figure 7A shows that an A2a antagonist enhances the ability of an A3/A1 agonist, MRS 580, to cause preconditioning. Figure 7B shows that an A2a antagonist enhances the protective effect of MRS 580 during prolonged ischemia. Figure 7C shows that MRS 580 can also precondition the cardiac myocyte via the human adenosine A3 receptor.

Figures 8A-8E are a series of graphs showing the cardioprotective effects of MRS1528, a binary conjugate having agonist activity at the A3 receptor and antagonist activity at the A2a receptors. Figure 8A shows that the ability of MRS1528 to cause preconditioning is not affected by the presence of 8-(3chlorostyryl)caffeine, (CSC). Myocytes were exposed to the indicated concentrations of MRS1528 in the presence or the absence of CSC (1 gM). Myocytes were also exposed to the indicated concentrations of MRS1528 in the presence or the absence of CGS21680 (0.3 gM). See Figure 8B. Figure 8C shows the results obtained when myocytes were exposed to MRS1525 in the presence or the absence of CSC. Figure 8D shows the results obtained when myocytes were exposed to the indicated concentrations of MRS1525 in the presence or the absence of CGS21680. The preconditioning effect of MRS1528 in cells transfected with the human adenosine A3 receptor is shown in Figure 8E. Data represent the means SE of three experiments.

Figure 9 is a diagram showing the synthetic scheme utilized to generate the binary compounds used in the WO 98/50047 PCT/US98/09031 practice of this invention.

Figures 10A-10C depict diagrams for synthesizing certain compounds of the invention. Figures 10A and show a synthetic scheme for synthesizing a derivative of an Al selective agonist for coupling to an amine derived A3 agonist. Figure 10C is a schematic diagram showing the synthesis of binary conjugates with extended linkers.

Figures 11A and 11B show the nucleotide sequence of the cDNA encoding the adenosine Al receptor.

Figures 12A-12D show the nucleotide sequence of the cDNA encoding the adenosine A2a receptor.

Figures 13A-13C show the nucleotide sequence of the cDNA encoding the adenosine A3 receptor.

DETAILED DESCRIPTION OF THE INVENTION The cardioprotective roles of adenosine Al and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and Cl-IB-MECA caused a dose-dependent reduction in the extent of hypoxia-induced injury, as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed.

The adenosine Al agonists CCPA and N6-cyclohexyladenosine (CHA) were also able to cause a decrease in the extent of myocyte injury. The Al WO 98/50047 PCT/US98/09031 receptor-selective antagonist DPCPX blocked the cardioprotective effect of the Al but not of the A3 agonists. Conversely, selective A3 antagonists MRS1191 and 3,5-Diethyl 2-methyl-6-phenyl-4- 2 -phenyl-(E)-vinyl]-1,4-(1)-dihydropyridine-3,5-dicarbo xylate (MRS 1097) blocked the protection induced by Cl-IB-MECA but had minimal effect on that caused by CCPA. Thus, the cardioprotective effects of Al and A3 agonists were mediated by their respective receptors.

The study identifies the cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that simultaneous activation of both Al and A3 receptors during hypoxia can attenuate myocyte injury. This finding is in contrast to that set forth by Downey et al. in U.S. Patent No. 5,573,772 wherein administration of antagonists to the Al receptor in conjuction with agonists at the A3 receptor was reported to enhance cardioprotective effects during ischemia.

Administration of an adenosine Al receptor antagonist is not required for practicing the present invention.

The present invention also includes administration of binary reagents that selectively activate the Al and A3 adenosine receptors simultaneously. Concomitant activation of the two receptors is believed to act synergistically to enhance the cardioprotective effects of preconditioning and to increase myocardial resistance to ischemia.

Several new MRS compounds are disclosed which selectively activate the A3 adenosine receptor. See Figures 3A-3F. These compounds are also contemplated for use in the methods of the present invention.

In accordance with another aspect of the invention, a binary conjugate has been developed, which binds both the A3 and A2a receptors simultaneously and elicits the desired result, activation of the A3 receptor and inhibition of the A2a receptor.

A second binary conjugate, has also been WO 98/50047 PCT/US98/09031 synthesized in accordance with this invention which simultaneously binds and activates both of the Al and the A3 receptors. These binary compounds may also be used to advantage in practicing the methods of the present invention. The following definitions are provided to facilitate understanding of the present invention.

Preconditioning ischemia A brief ischemia which does not cause any cardiac damage, but is able to protect the heart against damage during a subsequent prolonged ischemia. The effect of preconditioning ischemia is mediated by adenosine, which is released during the ischemia. Preconditioning may be induced by brief exposure to anoxic conditions for example.

Adenosine receptors Al, A3 and A2a receptors are present on the myocardium (cardiac muscle cells). While activation of the Al and A3 receptors is cardioprotective, activation of the A2a receptors is deleterious and causes damage to the cardiac muscle cells.

Stable angina Condition observed in certain cardiac patients having a chronic risk for mycardial ischemia because of the chronic potential for an imbalance between the supply of oxygenated blood and the demand for it. Typically, such imbalance occurs during certain stresses, such as exercise, emotional stress or stress associated with a surgical procedure.

Unstable angina Condition observed in cardiac patients having frequent imbalance between the supply of and the demand for oxygenated blood.

Post-myocardial infarction angina Condition observed in patients who have recurrent ischemia following a WO 98/50047 PCT/US98/09031 heart attack.

Preconditioning stimuli Any drug, agent or treatment which induces preconditioning, such as brief ischemia, or Al or A3 receptor agonists.

Myocardial responsiveness The myocardium can be treated so as to enhance the effectiveness and protective effects of preconditioning. This enhancement leads to a reduction in ischemic damage.

Representative examples of compounds used for practicing the present invention are as follows Al agonists CCPA, and compounds listed in Table I.

Al antagonists DPCPX A3 agonists IB-MECA, Cl-IB-MECA, MRS 584, MRS 537, MRS 479, MRS 1340, DBXMR, NNC21-0238, NN53-0055 A3 antagonists MRS 1191 A2a agonists CGS21680 A2a antagonists CSC Binary A3/A1 agonists MRS 1543 Binary A3 agonist/A2a antagonists MRS 1528 Mixed A3/A1 agonists compounds listed in Table II, including MRS 580 and MRS 1364 The present invention demonstrates that activation of adenosine receptors during prolonged hypoxia can attenuate the hypoxia-induced myocyte injury.

Specifically, activation of Al and A3 receptors has been shown to mediate adenosine-induced cardiac myocyte protection. Additionally, the concomittant activation of the A3 receptor coupled with inhibition of A2a receptor activation by a selective binary conjugate has also been shown to attenuate hypoxia-induced myocyte injury. The following methods facilitate the practice of the present invention.

Preparation of Cultured Ventricular Cells WO 98/50047 PCT/US98/09031 Ventricular cells were cultured from chick embryos 14 days in ovo as previously described (16, 23). Cells were cultivated in a humidified 5% C0 2 -95% air mixture at 37 0 C. All experiments were performed on day 3 in culture, at which time cells exhibited rhythmic spontaneous contraction. The medium was changed to a HEPES-buffered medium containing (mM) 139 NaC1, 4.7 KCl, MgC1 2 0.9 CaCl2, 5 HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 2% fetal bovine serum, pH 7.4, 37 0 C before exposing the myocytes to the various conditions at 370C. Control myocytes were maintained in the HEPES-buffered media under room air. Ninety-minute exposure of the myocytes to hypoxia with glucose deprivation was used to induce cell injury. Hypoxia was produced by placing the cells in a hypoxic incubator (NuAire) where 02 was replaced by

N

2 as previously described (16,23). Effects of adenosine receptor agonists and antagonists on the extent of myocyte injury were determined by exposure of the prepared cells to these agents during the prolonged hypoxia.

Determination of Cell Injury Determination of myocyte injury was made at the end of the ninety-minute hypoxia, at which time myocytes were taken out of the hypoxic incubator and re-exposed to room air (normal 02). Aliquots of the media were then obtained for creatine kinase (CK) activity measurement, which is followed by quantitation of the number of viable cells. Measurement of basal level of cell injury was made after parallel incubation of control cells under normal 02. The extent of hypoxia-induced injury to the ventricular cell was quantitatively determined by the percentages of cells killed and by the amount of CK released into the media according to a previously described method (16, 23).

Prior studies demonstrated that the cell viability assay WO 98/50047 PCT/US98/09031 distinguished the hypoxia-damaged from the control normoxia-exposed cells. In brief, the media were replaced with a trypsin-EDTA buffer to detach the cells, which was then followed by sedimentation of the viable myocytes. Parallel changes in cells killed and CK released (16, 23) further validated this assay for cells killed. The amount of CK was measured as enzyme activity (unit/mg), and increases in CK activity above the control level were determined. The percentage of cells killed was calculated as the number of cells obtained from the control group (representing cells not subjected to any hypoxia or drug treatment) minus the number of cells from the treatment group divided by number of cells in control group multiplied by 100%.

Synthesis of Adenosine Al and A3 Receptor-selective Agents N 6 (IB-MECA), 2-chloro-N 6 -(3-iodobenzyl)adenosine- (Cl-IB-MECA), and adenosine amine congener (ADAC) were synthesized as previously described (8,11,13). 3,5-Diethyl 2-methyl- 6-phenyl-4-[2-phenyl-(E)-vinyl]-1,4-(1)- (MRS 1097) and 3-ethyl 5-benzyl-2-methyl-6-phenyl- 4 -phenylethynyl-l,4-(1)-dihydropyridine-3,5dicarboxylate (MRS 1191) were synthesized as previously described (12).

Adenosine analogs 8 -cyclopentyl-l,3-dipropylxanthine (DPCPX) and 2-chloro-N 6 -cyclopentyladenosine

(CCPA),

N

6 -cyclohexyladenosine (CHA) were from Research Biochemicals International (Natick, MA). Embryonic chick eggs were from Spafas Inc. (Storrs, Conn).

WO 98/50047 PCT/US98/09031 Transfection of chick atrial myocytes with the human A3 receptor Atrial cells were cultured from chick embryos 14 days in ovo and transiently transfected with pcDNA3 (empty vector) or with pcDNA3/hA3R (full length cDNA encoding human adenosine A3 receptor subcloned in pcDNA3) using the calcium phosphate precipitation method. Forty-eight hours after transfection, the cells were exposed to the compounds of the invention and the percentage of myocytes killed during simulated ischemia was determined.

The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

EXAMPLE I ACTIVATION OF Al OR A3 ADENOSINE RECEPTORS REDUCES ISCHEMIC INJURY TO THE HEART A. Exogenous adenosine causes a decrease in the extent of cardiac myocyte injury during the prolonged hypoxia Prolonged exposure to hypoxia in glucose-free media induced significant cardiac myocyte injury with large increase in the release of creatine kinase (28.5 unit/mg, n= 4, SE) and in the cells killed (30 2 n= Adenosine (10 jM), when added to the media during the ninety-minute hypoxia, caused a decrease in the amount of CK released (CK released in unit/mg 14.9 3, n= 3) and in the cells killed cells killed= 12 2 n= This effect of exogenous adenosine was blocked by the nonselective adenosine receptor antagonist 8-sulfophenyl-theophylline (8-SPT, at 100 jM) (in the presence of adenosine and 8-SPT: CK released in unit/mg 31 5 and cells killed 28 3 The presence of 8-SPT during the hypoxia had no effect on the level of myocyte injury (CK released= 29.6 1.2 unit/mg; cells killed= 31.6 1.5 These data WO 98/50047 PCT/US98/09031 suggest that activation of adenosine receptors during the prolonged hypoxia can protect the myocyte against injury. Since adenosine can activate both the cardiac Al and A3 receptors and since 8-SPT, at 100 AM, can block both receptors the data are consistent with the hypothesis that either receptor or both receptor subtypes can mediate the cardioprotective function of adenosine. This hypothesis was further explored using selective agonists and antagonists. See Figures 1, 2 and 3.

B. Adenosine A3 agonists mediate cardioprotective effects during prolonged hypoxia In order to examine whether activation of adenosine A3 receptors is capable of attenuating myocyte injury during the prolonged hypoxia, agonists selective at the A3 receptor were used. Prior study has demonstrated that both IB-MECA and Cl-IB-MECA are highly selective at the chick cardiac A3 receptor Either A3 agonist, when present during the prolonged hypoxia, was capable of protecting the cardiac myocytes against hypoxia-induced injury (Figure The cardioprotective effect of A3 agonists was quantitated as a decrease in the amount of CK released and the percentage of myocytes killed (statistically significant at 1 and 10 nM of Cl-IB-MECA, ANOVA and t test, p<0.01).

The presence of the Al receptor-selective antagonist DPCPX had no effect on the ability of IB-MECA or Cl-IB-MECA to mediate their cardioprotective effects; the A3 agonist-induced decreases in cells killed and CK released were similar in the presence and absence of 1 AM of DPCPX (not shown). These data indicate that the cardioprotective effect of the A3 agonists was not due to activation of the cardiac Al receptor.

To determine whether the A3 agonist-induced cardioprotection is mediated by the A3 receptor, antagonists selective at the A3 receptor were employed.

WO 98/50047 PCT/US98/09031 Figure 1 demonstrates that the A3 receptor-selective antagonist MRS 1191 blocked the Cl-IB-MECA-induced cardioprotection. The levels of CK released and of percentage of cells killed were significantly higher in myocytes exposed to Cl-IB-MECA and 30 nM, 300 nM or 3 .M of MRS 1191 (ANOVA and t test, p<0.01). Another A3 receptor antagonist, MRS1097, was also able to block the cardioprotective effect of Cl-IB-MECA (not shown).

These data provide conclusive evidence that activation of the A3 receptor can produce a potent cardioprotective effect when administered during the prolonged hypoxia.

C. Adenosine Al receptor activation reduces cardiac myocyte injury during prolonged ischemia Since the Al receptor is also present on the cardiac myocyte, an investigation was undertaken to determine whether activation of the Al receptor can confer a cardioprotective effect during the prolonged hypoxia. Prior study showed that CCPA, a known Al agonist, is highly selective at the Al receptor on these cardiac myocytes Cultured ventricular myocytes were prepared and the extent of hypoxia-mediated myocyte injury determined. The various adenosine Al receptor agonists were added to the media at the indicated concentrations in the absence or the presence of the Al receptor antagonist DPCPX during the prolonged hypoxia.

The percentage of cells killed was determined following the hypoxic exposure and removal of the Al receptor agonists and antagonist. The data represent the mean of four experiments. At 1 and 10 nM of the Al agonists, the percentages of myocytes killed were significantly lower than those obtained in the presence of either of the two Al agonist concentrations and DPCPX (1 gM) (ANOVA and t test, P<0.01). Thus, CCPA caused a dose-dependent reduction in the percentage of myocytes killed as shown in Figure 2 and in the amount of CK released (not shown) (ANOVA and t test, p<0.01). Two WO 98/50047 PCT/US98/09031 other Al receptor-selective agonists, ADAC and CHA, were also able to protect the myocytes when present during the prolonged hypoxia. The cardioprotection stimulated by CCPA, CHA and ADAC was blocked by the Al antagonist DPCPX. On the other hand, neither MRS 1191 nor MRS 1097 was able to block the CCPA-induced cardioprotection as shown in Figure 1, providing definitive evidence that the Al agonist effect is mediated by the Al receptor.

Although the number of viable cells was determined quickly following re-exposure of cardiac myocytes to normal 02 (reoxygenation), the Al or the A3 agonist was nevertheless present briefly prior to replacement with the trypsin-EDTA buffer for cell viability assay. Thus, it is possible that the decrease in myocyte injury is due to the protection against a reoxygenation injury.

To study this possibility, CCPA or Cl-IB-MECA was added immediately upon reoxygenation following the ninety-minute hypoxic exposure. CCPA or Cl-IB-MECA was maintained in the media for an additional hour prior to determination of the percentage of myocytes killed.

Although CCPA or Cl-IB-MECA was able to protect the myocytes when present during the reoxygenation, the extent of protection was small myocytes killed following the 90 minute hypoxia= 26.5 1.0 n=6, S.E. vs. CCPA present= 22.1 1.5 n=5 or vs.

Cl-IB-MECA present= 23.0 1.4 n=5; ANOVA and t test, P<0.05) The previous example illustrates the efficacy of A3/A1 adenosine receptor agonists in reducing ischemic damage to the heart. A variety of other compounds have been developed which also stimulate WO 98/50047 WO 9850047PCT/US98/09031 A3 adenosine receptors.

These are set forth below: MRS 584

(CH

2 3

CH

3 0 N

N)

/N

N

0H 3 (0H 2 3 0 0 11 0 MRS 479 WO 98/50047 WO 9850047PCTIUS98/09031

CH

3

NHN

0

N

CH

3 NHC, 0 HO OH MRS 537 S0 3

H

NHCH

2

NN

N

NN

CH

3 NHC 0 HO OH MRS 1340 WO 98/50047 PCT/US98/09031 Additional A3 selective agonists are set forth below:

A

s selectlve >w jj N H R H, IB.MECA N N YN r nWWIS C1HNHC 0 0CH,(CH) R= CKC 2 R CI, CIB-MECA 0 0 cHN N M 8204700.33 o 1230/l,4000.(h) 82M70/3 4CHNHCO 0 R CHOCH 3 HO OH NNC53-0088 37 ,S00=OWM 9 O HO OH 100B5004.8(h) The compounds shown immediately above have been assayed for cardioprotective efficacy during prolonged ischemia in the in vitro culture system described in Example I.

The cardioprotective effects of A3 receptor agonists MRS 584, MRS 537, MRS 479, MRS 1340 were assessed in the presence or absence of the Al receptor antagonist DPCPX. The Al receptor antagonist was utilized to demonostrate the selectivity of the MRS compounds for the A3 as opposed to the Al adenosine receptor. Figure 3A shows the cardioprotective effects of MRS 584 in the presence and absence of DPCPX. The extent of myocyte injury was plotted as the amount of creatine kinase released during the prolonged simulated ischemia. Figure 3B shows the cardioprotective effects of MRS 537 in the presence and absence of DPCPX. The WO 98/50047 PCT/US98/09031 extent of myocyte injury was plotted as the amount of creatine kinase released during the prolonged simulated ischemia as a function of increasing the concentrations of MRS 537. Figure 3C shows the cardioprotective effects of MRS 479 in the presence and absence of DPCPX.

The extent of myocyte injury was plotted as the amount of creatine kinase released during the prolonged simulated ischemia as a function of increasing the concentrations of MRS 479. Figure 3D shows the cardioprotective effects of MRS1340 in the presence and absence of DPCPX. The extent of myocyte injury was plotted as the amount of creatine kinase released during the prolonged simulated ischemia, as a function of increasing the concentrations of MRS1340.

To determine whether the protective effects of the compounds of the invention would also comparably stimulate the human adenosine A3 receptor, atrial cardiac myocytes, which express little if any endogenous A3 receptor, were transfected with either empty vector or vector containing the human adenosine A3 receptor cDNA. The transfected myocytes were then exposed to the compound for 5 minutes, which was followed by replacement with fresh media and myocytes exposed to normal conditions (non-ischemic) for 10 minutes prior to being exposed to ninety minutes of ischemia. The decrease in the number of myocytes killed in A3 receptor cDNA-transfected myocytes as compared to vector-transfected myocytes was plotted as a function of the concentration of the compound. This decrease in myocytes killed represented an index of the protection mediated via the human adenosine A3 receptor.

Figure 3E shows the protective effects of MRS 584 and MRS 537 obtained when chick cells were transfected with a human A3 receptor encoded cDNA. These data show that the MRS compounds of the invention specifically bind to and activate the human A3 receptor.

Figure 3F shows that chick atrial cells aquire A3- WO 98/50047 PCT/US98/09031 receptor mediated cardioprotective responses upon transfection with human A3 receptor encoding cDNA.

Cells were exposed to 10 nM Cl-IB-MECA and 1 pM DPCPX for five minutes. Media were replaced with fresh media lacking Cl-IB-MECA or DPCPX. Cells were then incubated under room air for thirty minutes prior to being exposed to 90 minutes of simulated ischemia. The graphs shows the results obtained in cells transfected with A3 receptor encoding cDNA, untransfected cells and cells transfected with empty vector. In untransfected cells or cells transfected with empty vector, the A3 agonist was able to reduce the percentage of cells killed when compared to cells not pre-exposed to A3 agonist (control cells, t-test P<0.05). However, the A3 receptor mediated reduction in the number of cells killed or the amount of CK released, expressed as decrease from those obtained in the control cells, was significantly greater in the cells transfected with the human A3 receptor encoding cDNA than in untransfected or cells transfected with empty vector (one-way ANOVA and t test P<0.01**) These data illustrate that these compounds selectively activate human adenosine A3 receptor and can be used to prevent ischemic damage to the heart.

Each of the above described MRS compounds is contemplated for use in combination with any of the Al agonists described herein for reducing ischemic damage to the heart.

EXAMPLE II ADENOSINE Al AND A3 RECEPTOR AGONISTS ACT SYNERGISTICALLY TO INDUCE CARDIOPROTECTION.

Adenosine can exert two principal cardioprotective effects. Adenosine can precondition the heart with reduction in the size of myocardial infarction (4,5,9,14,17,18). Intracoronary administration of WO 98/50047 PCT/US98/09031 adenosine during reperfusion following prolonged no-flow ischemia can also limit infarct size .in the intact heart 19). Our previous studies have characterized a cardiac myocyte model of injury, which is induced by exposure of myocytes to a prolonged period of hypoxia in glucose-free media (16, 23). Activation of either the Al or the A3 receptor by their respective agonists can pharmacologically precondition the cardiac myocyte (23).

Figures 1 and 2 show that the presence of Al or A3 agonist can also protect the cardiac myocytes when the agonist is present individually during an actual injury-inducing ischemia.

Figure 4A shows that the simultaneous activation of both Al and A3 receptors produces a greater preconditioning effect than when either receptor is activated separately. Thus, an Al agonist and an A3 agonist interact synergistically to precondition the cardiac myocyte. In addition, the simultaneous presence of Al and A3 receptor agonists during injury-producing ischemia resulted in less myocyte injury than when only Al or A3 agonist is present. See Figure 4B. Thus, simultaneous administration of Al and A3 agonists will lead to enhanced cardioprotection.

Based on the data presented above, it is apparent that a variety of Al agonists and A3 agonists may be used in conjunction to reduce or prevent ischemic damage to the heart. Suitable Al agonists contemplated for use in the present invention are set forth below in Table I.

WO 98/50047 WO 9850047PCTIUS98/09031 TABLE I Al AGONISTS A, selective HOOH(~0 059/460/240 R CCPA HO OH O*6/9,5q1237

ADAC

0.85/210/281

R-PIA

1-21124158 H3S NH HOCH W

SPA

HO OH R s/IA I 3/51017 R CH 3 SDZ WAG 994 7615700/-

-H

3 NNC 21-0136 11/6601- WO 98/50047 PCT/US98/09031 EXAMPLE III CARDIOPROTECTIVE EFFECT OF AGONISTS ACTIVATING BOTH Al AND A3 RECEPTORS In yet another embodiment of the invention, the administration of an agonist capable of activating both Al and A3 receptors will be used in the methods of the invention for reducing ischemic damage to the heart.

Referred to herein as a mixed A3/A1 agonist, these agents mediate superior cardioprotective effects.

Examples of mixed A3/A1 agonists that may also be used in the practice of the present invention are listed in Table II.

NHR

NN

0 0N

H

HO OH WO 98/50047 WO 9850047PCT/US98/09031 R, R Al Az.(nm) A 3 COMPOUND (nm) (nm) (Reference) Compound 4q Ethyl 116 3940 (Reference 27) H MRS 646 c Compound 4d Ethyl F 3 C 384 >10,000 54 (Reference 27) H

N

Compound 37 Ethyl -49 574 (Reference 8) NO2 Compound 11 Methyl 0 2060 66,300 1340 (Reference 28)

N

6 -cyclohexyl Ethyl 0.43 170 16 NECA (Reference 29) 4q N 6 (3-iodophenyl )carbamoyl )adenosine uronamide 4d N 6 (2-trifluoromethyl )carbamoyl) adenosine-5' uronamide compound 37 N 6 (4-Nitrobenzyl) adenosine-5 '-N-ethyluronamide compound 11 (0-Phenylhydroxylamino)purine-9-beta-ribofuranoside- -N-methyluronamide N6-cyclohexyl NECA N6-cyclohexyl WO, 98/50047 WO 9850047PCTIUS98/09031 COMPOUND R=A, (nM) A, (nM) A 3 (nM) Compound 8' 2-aminoethyl)amino] 0.85 210 4 carbonyl ]methyl anilino] carbonyl ]methyl] phenyl *N 6 [[4-[2-alninoethyl)amino]carborlyl]methyl]anilino] carbonyl]methyl ]phenyl] adenosine Other examples of mixed Al/A3 agonists are MRS 580 and MRS 1364, the structures of which are shown below.

WO 98/50047 MRS 580 PCTIUS98/0903 1 0 K0

CH

3 -N *0NNF OH

OH

NHR

N~ ~N N0

N

R'=Et R CONH-phenyl-NO 2

H

MRS 1364 WO 98/50047 PCT/US98/09031 Figure 5A shows the results obtained when ventricular myocytes were exposed to the indicated concentrations of MRS646 (structure 4Q, table II) or MRS1364 for 5 minutes. Media were then replaced with fresh media, which was then followed by exposure to simulated ischemia and the data graphed. The data show that these mixed A3/A1 agonists can pharmacologically induce preconditioning in cardiac myocytes.

Figure 5B shows that MRS 646 and MRS 1364 also potently protect against myocyte injury during the sustained simulated ischemia. Figures 5C and 5D show that these agonists can pharmacologically precondition the cardiac myocyte and induce potent cardioprotection via the human adenosine A3 receptor.

In yet another embodiment of the invention, a binary conjugate has also been synthesized which binds and activates both the Al and A3 receptors simultaneously. Figure 6A shows that this binary conjugate, MRS1543, can pharmacologically precondition the cardiac myocyte and induce a potent cardioprotective effect. The protective effect is only partially blocked by Al antagonist DPCPX; similarly, the protection is only partially attenuated by the A3 antagonist MRS1191.

Figure 6B shows, however, that the combined presence of DPCPX and MRS1191 completely abolished the protective effect of MRS1543, indicating that the protection is mediated via activation of both the Al and the A3 receptors. Figure 6C shows that MRS1543 can precondition and induces a cardioprotective effect via the human adenosine A3 receptor.

WO 98/50047 PCT/US98/09031 EXAMPLE IV SIMULTANEOUS ADMINISTRATION OF A3/A1 ADENOSINE RECEPTOR AGONIST AND ADENOSINE A2a RECEPTOR ANTAGONIST GIVES RISE TO ENHANCED CARDIOPROTECTION In yet another embodiment of the invention the simultaneous administration of A3 and Al adenosine receptor agonist and A2a antagonist is contemplated.

Preferred A2a adenosine receptor antagonists for use in the present invention are listed below in Table III: R1, R3 methyl, ethyl, propyl, allyl R7 H, methyl, alkyl (C2-C8) Ra H (unless noted) X is shown in Table III WO 98/50047 WO 9850047PCTIUS98/09031 TABLE III Affinities of 8 Styrylxanthine Derivatives in Radioligand Binding Assays at Rat Brain A, and A,-Receptors (31) cmpd R,.R 3 R7X A YAi ratio 15b l7b 19b 21b 22b 23 24 26 27b 28 29b 32b M3a 33b 34b 36 37 38 39 41 42 43b 44b 46 Sib 52b 11 2-MeO 3-MeO 3-CF., 3-NO, 3-NII, 3-(AcNII) 3-(IIOOC(C1 2

),CONII)

3-0-IIOC-NII) 3--IIC) 2

N]

3-F 3-Cl 4-MeO 3.4-(MeO), 3.5-(MeO), 3.5-(NteO), 3.5-F, 3.5-(MeOh2-4-011 4-AcO-3.S-(MeO), 4-(4-P'hCI 1 1 0)-3,5-(MeO), 4-(4-N11 1 -lluO)-3.5 (MeO) 2 4-14-QIIIOC-NI)BtuOI-3,5-(MeO), 4-(4-Nl1-irans-CII 2 ClH= CIICI1 2 0-3,5-(MeO), 4-(4-AcNII-lirans-CII 2 Cl-l= ClI1ll0),54(MO), 4-(4-i-BOC-NII-lrans-CHICH= CIC1110-3,5-(MeOX, 2,3.4-(MeO), 3.4.5-(MeO), 3.4.5-(MeO), 3,4.5-(rMeO), 3-Cl 3.4-(MeO), 3.5-(N4eo) 2 41 18 64 11 240 250 is 190 520 44 >200 230 19 93 36 42 28 34 1 >56001 34 13 1 >6700] 14 19 [1901 110 53b Pr WO 98/50047 WO 9850047PCTIUS98/09031 Additional compounds contemplated for use as A 2 a antagonists include: H C0

CH

2

HC~~

2 C N N Y rn-Br or p-SO 3 H (DMPX)

NH

2 HO \'N N N

H

(ZM24 1385) (SCH582 61) WO 98/50047 PCT/US98/09031 The results shown in Figures 7A and 7B indicate that the simultaneous administration of an A3/A1 agonist and an A2a antagonist gives rise to enhanced cardioprotection. Cardiac myocytes were prepared as described in Example I. The A1/A3 agonist, MRS 580, was delivered in the presence or absence of the A2a antagonist CSC. Data were plotted as the percentage of cells killed vs. the various drug combinations as indicated. Data represent the means S.E. of three experiments. Figure 7C shows that MRS 580 can precondition the cardiac myocytes via the human adenosine A3 receptor.

In another embodiment, a binary conjugate, MRS 1528, was synthesized which acts as an agonist at the A3 receptor and an antagonist at the A2a receptor simultaneously. Figure 8A shows that the protective effect of MRS1528 was unaffected by the A2a receptor antagonist CSC, consistent with the ability of MRS1528 to block the A2a receptor. Figure 8B shows that the protective effect of low concentrations of MRS1528 is attenuated by the A2a agonist CGS21680 whereas the protective effect of high concentrations of MRS1528 is not affected by the presence of CGS21680. Together, these data indicate that MRS1528 can activate A3 receptor to induce preconditioning and can simultaneously block the A2a receptor to enhance its preconditioning effect.

Further support for this concept comes from studies testing the A3 agonist moiety of MRS1528, MRS1525.

MRS1525 does not contain the A2a antagonist moiety and in response to the CSC, showed a uniform CSC-mediated increase in the extent of preconditioning effect (Fig.

8C). In the concomitant presence of CGS21680, the preconditioning effect of MRS1525 is attenuated at both the high and low concentrations of MRS1525 (Fig. 8D).

Figure 8E shows that MRS1528 can cause preconditioning of WO 98/50047 PCT/US98/09031 the cardiac myocytes via human adenosine A3 receptor.

A binary conjugate was synthesized with the general structure shown below. This conjugate binds both the A2a and the A3 receptors and acts as an agonist at the A3 receptor and an antagonist at the A2a receptor simultaneously. In the exemplary conjugate disclosed herein, MRS 1528, R H and n =2.

A3/A2a binary conjugate CF3 0 N/CH3 C CCH 2 NHC(CH2)CONHO

N

N

CH3 n=2-4 A3/A2a Binary conjugate R H, IB-MECA; R Cl, C1-IB-

MECA

WO 98/50047 PCT/US98/09031 A second binary conjugate has also been synthesized which binds and activates both the Al adenosine and A3 adenosine receptors simultaneously and has the general structure shown below. In an exemplary A1/A3 binary conjugate of the invention, MRS 1543, R=H.

C =CCHNHCOCH, -NHCOCH 2

NH

N/ N NH CH, R N HOCH, 0 R -NH

CH

3 NHC ,0 HO HO OH A3/A1 binary conjugate R H, IB-MECA R Cl, CL-IB-MECA Figure 9 sets forth an exemplary synthetic scheme utilized to produce the binary conjugate specific for the Al and A3 receptors.

Figures 10A -10C depict schematic diagrams for synthesizing the compounds of the invention. Figures 10A-10B shows a synthetic scheme for generating a derivative of an Al selective agonist for coupling to an amine derived A3 agonist. Figure 10C shows a synthetic scheme for conjugating the reagents via an extended linker. Extended linkers may increase the affinity and potency of the conjugates at the adenosine receptor.

Table IV lists the names of the chemical structures appearing in Figures 9, 10B and WO 98/50047 WO 9850047PCTIUS98/09031 TABLE IV IB-MECA =N 6 methylcarboxamidoadenosine Compo~und III N 6 -[3-(3-amino-l-propynyl)benzyl]-5'- N-xnethylcarboxamidoadenosime Compound VIII and p-isomers) N 6 isothiocyanatophenylaminothiocarbonyl) -amino-imethylcarboxamidoadenosime N-and N-3- (4-isothiocyanatophenylaminothiocarbonyl) amino-l-propynyl ]benzyl] -Nmethylcarboxamidoadenosine ADAC N 6 [4-fi[[4-rrrc:2aminoethyl) amino~carbonyl]methyl] anilino]carbon yl ]methyl ]phenyl ]adenosine 206 Compound IX (in- and p-isomers) =conjugate of N 6_ [[(2-aminoethyl)aiinocarbonyl~methyl anilino)carbonyllme thyl]phenyl]adenosine and N isothiocyanatophenylaminothiocarbonyl) -amino-ipropynyl] benzyi] methyicarboxamidoadenosine a n d6 4 2 a i o conjugate of N 6 ethyl) amino]carbonyl]methy1 anilino]carbony)ne thyi~phenyl~adenosine and N isothiocyanatophenylaninothiocarboiyl) -amino-ipropynyl ]benzyl] methylcarboxamidoadenosine Compounds XI= MRS 1576 t-Boc-L-alanyl-N 6 _[3-(3-amino-lpropynyl) benzyl) iethylcarboxamidoadenosine MRS 1574 =t-Boc-L-methionyl-N6-[3--(3-amino-1propynyl) benzyi] methyicarboxainidoadenosime MRS 1568 t-Boc-L-vaiyi-N 6 -[3-(3-amino-1propynyl) methylcarboxamidoadenosime MRS 1571= t-Boc-L-leucyl-N 6 _-3-(3-amino-lpropynyl) benzyl] methylcarboxainidoadenosime MRS 1572 t-Boc-L-isoleucyl-N 6 [3-(3-amino-l WO 98/50047 WO 9850047PCTIUS98/09031I methylcarboxamidoadenosime MRS 1573 t-Boc-L-phenylalanyl-N 6 _(3-.(3-amino-1propynyl) benzyl] methylcarboxamidoadenosine Compounds XII= MRS 1577 L-alanyl-N 6 -[3-(3-amino-lpropynyl) benzyl] -Nmethylcarboxamidoadenosime MRS 1575 L-methionyl-N 6 -[3-(3-amino-lpropynyl) methylcarboxamidoadenosime MRS 1570 L-valyl-N 6 -[3-(3-amino-lpropynyl) benzyl] methylcarboxamidoadenosine MRS 1569= L-leucyl-N 6 [3-(3-amino-lpropynyl) benzyl] methylcarboxamidoadenosine MRS 1560= L-isoleucyl-N 6 -[3-(3-amino-lpropynyl) benzyl] -Nmethylcarboxamidoadenosine MRS 1565 L-phenylalanyl-N 6 -[3-(3-amino-lpropynyl) benzyl] methylcarboxamidoadenosime Compound IV 64[C4 (carboxymethyl) anilino]carbonyl ]Iethyl]phenyl] a denosine Compound XIII =conjugate ofN6_[-[4 (carboxymethyl) anilino]anilino~carbonyl]methyl] phenyl) adenosine and compound XII Compound XIV N 6 _(4-(carboxymethyl)phenyl]adenosine Compound XV conjugate of N6-[4- (carboxymethyl) phenyl] adenosine and compound XII WO 98/50047 PCT/US98/09031 EXAMPLE V SCREENING ASSAY FOR IDENTIFYING COMPOUNDS THAT HAVE AFFINITY FOR THE A3 ADENOSINE RECEPTOR.

The recombinant cardiac atrial cells described above provide a system for assessing agents that may have cardioprotective activity. A generalized method for such screening would entail providing a fixed concentration of a test compound to transfected cells and assessing whether or not the test compound exerted a protective effect during ischemia. Negative controls would comprise both untranfected cells and transfected cells not exposed to the test compound.

Once a test compound is determined to have cardioprotective activity, it will be serially diluted and applied to the transfected myocytes described above.

In this way, the minimally effective concentration of the compound will be determined.

While CaPO 4 transfection is exemplified herein, the myocytes of the invention may be transfected using any method known to those of skill in the art. Such methods include, but are not limited to lipofectin, electroporation, or viral vector mediated transfection.

Cardiac myocytes may be transfected with any of the adenosine receptor having a known DNA sequence. Thus the assay is not limited to cells transfected with A3 encoding cDNA. Any of the known adenosine receptors may be transfected and assayed as described above. Figure 11 shows the nucleotide sequence of the cDNA encoding the Al adenosine receptor. Figure 12 shows the nucleotide sequence of the cDNA encoding the A2a receptor and Figure 13 shows the sequence of the cDNA encoding the A3 receptor (32-34).

WO 98/50047 PCT/US98/09031 EXAMPLE VI ADMINSTRATION MODALITIES SUITABLE FOR THE COMPOUNDS OF THE PRESENT INVENTION The protective effect of A3/A1 agonists has been demonstrated herein in animal models. A1/A3 agonists may be used therapeutically in patients who suffer from ischemic damage due to stable angina, unstable angina or post-myocardial infarction angina.

Several administration modalities may be utilized to treat patients with the agonists and antagonists of the invention. These modalities are influenced by bioavailability factors. For example, if the compound is metabolized in the liver or excreted in the bile, some of the active compound absorbed from the gastrointestinal tract will be inactivated by the liver before it can reach the general circulation for distribution to the site of action. It is not believed that the compounds of the invention will be subject to this first pass loss.

Additionally, because the agonists of the invention are polar and water soluble, it is expected that they will have a small volume of distribution, and thus be readily eliminated by the kidney. Moreover, binding of the agonists to plasma proteins may limit their free concentrations in tissues and at their locus of action since it is only the unbound drug which equilibrates across membrane receptor sites.

Another factor affecting bioavailability is the distribution of the agonists to tissues. The agonists of the invention do not cross the blood brain barrier.

Given the relatively small size of the compounds and their water solubility, it is anticipated that the compounds will have a relatively fast second phase of drug distribution. This distribution is determined by both the blood flow to the particular tissue of the organ such as the heart, as well as the rate at which the compounds diffuse into the interstitial compartment from WO 98/50047 PCT/US98/09031 the general circulation through the highly permeable capillary endothelium.

Patients may be perfused with the agonists of the invention by dissolving them in normal saline solution or using emulsifying agents or cosolvents followed by intravenous administration every four to six hours.

Effective doses usually range from 100 to 300 nM. For example, considering a 15 liter volume of distribution for a 70 kg patient, a loading dose ranging from 0.5 to 1.5 mg is preferably used. Depending on the half-life of the agonists in the body, several doses, 1.5-4.5 mg may be adminstered per day.

Alternatively, a time-release or slow-release preparation may be utilized which allows for periodic or constant release of the antagonists over a given time period. This method would allow for a single dose of the agonists in a given day. Methods for preparing such capules are well known to those of skill in the art of drug delivery.

In summary, the present data illustrates the novel synergistic protective function of simultaneous cardiac Al and A3 receptor activation. In addition to the synergistic role in mediating preconditioning of the cardiac myocyte, the data provide conclusive evidence that activation of both receptors can also act synergistically to attenuate myocyte injury during the prolonged injury-producing ischemia. Thus, agonists selective at the Al and the A3 receptors represent novel potent cardioprotective agents even when ischemia has already begun. The concomitant administration of A1/A3 agonist with an A2a antagonist may also enhance cardioprotection as will binary conjugate capable of activating the Al or A3 receptor while simultaneously blocking the A2a receptor. These data have important clinical implications in the treatment of ischemic heart disease and implicate Al and A3 receptor-selective agonists for the reduction of the size of myocardial WO 98/50047 PCT/US98/09031 infarction when given during the infarct-producing ischemia.

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While certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made to the invention without departing from the scope and spirit thereof as set forth in the following claims.

Claims (54)

1. A method for preventing or reducing ischemic damage to the heart, in a patient in need thereof, comprising administering to said patient an agonist having affinity for both the Al and A3 adenosine receptors in an amount effective to activate A3 and Al receptors in the heart of said patient.

2. A method as claimed in claim 1, wherein said agonist is delivered using an administration means selected from the group consisting of intravenous administration, oral administration and cardiac perfusion.

3. A method as claimed in claim 1, wherein said agonist is selected from the group of compounds listed in Table II.

4. A method as claimed in claim 1, wherein said agonist is N 6 -((2-trifluoromethyl)carbamoyl) A method as claimed in claim 1, wherein said agonist is N 6 -((3-iodophenyl)carbamoyl)

6. A method as claimed in claim 1 wherein said agonist is a binary conjugate which has affinity for, and activates the Al and A3 adenosine receptors simultaneously.

7. A method as claimed in claim 1 wherein said agonist is administered to said patient prior to a surgical procedure having potential to cause cardiac ischemic damage. WO 98/50047 PCT/US98/09031

8. A method as claimed in claim i, wherein said agonist is administered to said patient during a surgical procedure having potential to cause cardiac ischemic damage.

9. A method as claimed in claim 1, wherein said agonist is administered to said patient following a surgical procedure having potential to result in cardiac ischemic damage. A method as claimed in claim i, wherein said patient is in need of said treatment due to an anginal condition selected from the group consisting of chronic stable angina, unstable angina, post myocardial infarction angina.

11. A method as claimed in claim i, wherein said patient is in need of such treatment due to acute myocardial infarction.

12. A method for preventing or reducing ischemic damage to the heart, in a patient in need thereof, comprising administering to said patient an mixed agonist having affinity for the A3 and Al adenosine receptors and an antagonist having affinity for the A2a adenosine receptor in amounts effective to activate said A3 and Al receptors and inhibit activation of said A2a receptor in the heart of said patient.

13. A method as claimed in claim 12, wherein said agonist and said antagonist are delivered using an administration means selected from the group consisting of intravenous administration, oral administration and cardiac perfusion.

14. A method as claimed in claim 12, wherein said agonist is selected from the group of compounds listed in Table II. A method as claimed in claim 12 wherein said antagonist is selected from the group of compounds listed in Table III.

16. A method as claimed in claim 12, wherein said agonist is N6-((2-trifluoromethyl)carbamoyl)adenosine-

17. A method as claimed in claim 12, wherein said agonist is N6-((3-iodophenyl)carbamoyl)adenosine-5'uronamide.

18. A method as claimed in claim 12, wherein said agonist as hereinbefore described is selected from the group consisting of MRS 584, MRS 479, MRS 537 or MRS 1340. .19. A method as claimed in claim 12, wherein said antagonist as hereinbefore described is selected from the 20 group consisting of CSC, DMPX, ZM241385 or SCH58261.

20. A method as claimed in claim 12, wherein said agonist and said antagonist are administered to said patient prior to a surgical procedure having potential to cause cardiac ischemic damage.

21. A method as claimed in claim 12, wherein said agonist and said antagonist are administered to said patient during a surgical procedure having potential to cause cardiac 30 ischemic damage.

22. A method as claimed in claim 12, wherein said agonist and said antagonist are administered to said patient following a surgical procedure having potential to result in cardiac ischemic damage. WO 98/50047 PCT/US98/09031

23. A method as claimed in claim 12, wherein said patient is in need of said treatment due to an anginal condition selected from the group consisting of chronic stable angina, unstable angina, and post myocardial infarction angina.

24. A method as claimed in claim 12, wherein said patient is in need of said treatment due to acute myocardial infarction. A method for preventing or reducing ischemic damage to the heart, in a patient in need thereof, comprising administering to said patient a binary conjugate, which acts as an adenosine A3 receptor agonist while simultaneously inhibiting the activation of A2a receptors in an amount effective to enhance myocardial response to said preconditioning stimuli.

26. A method as claimed in claim 25, wherein said patient is in need of such treatment due to a cardiac condition selected from the group consisting of chronic stable angina, unstable angina, post-myocardial infarction angina or acute myocardial infarction.

27. A method as claimed in claim 25 wherein said agonist is administered to said patient prior to a surgical procedure which may cause cardiac ischemic damage.

28. A method as claimed in claim 25, wherein said agonist is administered to said patient during a surgical procedure having potential to cause cardiac ischemic damage.

29. A method as claimed in claim 25, wherein said agonist is administered to said patient following a surgical procedure which may result in cardiac ischemic WO 98/50047 PCT/US98/09031 damage. A method for preventing or reducing ischemic damage to the heart, in a patient in need thereof, comprising administering to said patient both an adenosine A3 receptor agonist and at least one adenosine Al receptor agonist in an amount effective to activate the Al and A3 adenosine receptors in the heart of said patient.

31. A method as claimed in claim 30, wherein said agonists are delivered using an administration means selected from the group consisting of intravenous administration, oral administration and cardiac perfusion.

32. A method as claimed in claim 30, wherein said agonist and said agonists are administered to said patient prior to a surgical procedure having potential to cause cardiac ischemic damage.

33. A method as claimed in claim 30, wherein said agonist and said antagonist are administered to said patient during a surgical procedure having potential to cause cardiac ischemic damage.

34. A method as claimed in claim 30, wherein said agonist and said antagonist are administered to said patient following a surgical procedure having potential to result in cardiac ischemic damage. A method as claimed in claim 30, wherein said patient is in need of said treatment due to an anginal condition selected from the group consisting of chronic stable angina, unstable angina, and post myocardial infarction angina. WO 98/50047 PCT/US98/09031

36. A method as claimed in claim 30, wherein said patient is in need of said treatment due to acute myocardial infarction.

37. A method as claimed in claim 30, wherein said A3 agonist is selected from the group of compounds consisting of IB-MECA, CI-IB-MECA, MRS 584, MRS 479, MRS 537, MRS 1340 and DBXMR and said Al agonist is selected from the group of compounds listed in Table I.

38. A binary conjugate for preventing or reducing ischemic damage to the heart, said conjugate acting as an agonist at the A3 adenosine receptor and an antagonist at the A2a adenosine receptor.

39. A binary conjugate as claimed in claim 38, said conjugate having the structure of MRS 1528.

40. A method for administering the binary conjugated of claim 38, wherein said conjugate is delivered using an administration means selected from the group consisting of intravenous administration, oral administration and cardiac perfusion.

41. A method as claimed in claim 40, wherein said conjugate is administered to said patient prior to a surgical procedure having potential to cause cardiac ischemic damage.

42. A method as claimed in claim 40, wherein said conjugate is administered to said patient during a surgical procedure having potential to cause cardiac ischemic damage.

43. A method as claimed in claim 40, wherein said conjugate is administered to said patient following a WO 98/50047 PCT/US98/09031 surgical procedure having potential to result in cardiac ischemic damage.

44. A method as claimed in claim 40, wherein said patient is in need of said treatment due to an anginal condition selected from the group consisting of chronic stable angina, unstable angina, and post myocardial infarction angina.

45. A method as claimed in claim 40, wherein said patient is in need of said treatment due to acute myocardial infarction.

46. A binary conjugate for preventing or reducing ischemic damage to the heart, said conjugate acting as an agonist at the A3 adenosine receptor and an agonist at the Al adenosine receptor.

47. A binary conjugate as claimed in claim 46, said conjugate being selected from the group of compounds consisting of MRS1543, a conjugate of N 6 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' on compound XII is H, a conjugate of N 6 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' on compound XII is Ch 3 a conjugate of N 6 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' is (CH 2 2 SCH 3 a conjugate of N 6 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' is CH(CH 3 2 a conjugate of N 6 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' is CH 2 CH(CH 3 2 a conjugate of N 6 51 WO 98/50047 PCT/US98/09031 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' is C(CH 3 )CH 2 CH 3 and a conjugate of N 6 (carboxymethyl)anilino]anilino]carbonyl]methyl]phenyl] adenosine and compound XII of Figure 10B, wherein R' is CH 2 C 6 H 5

48. A binary conjugate as claimed in claim 46, said conjugate being selected from the group of compounds consisting of a conjugate of N6-[4-(carboxymethyl) phenyl]adenosine and compound XII of Figure 10B, wherein R' is H, a conjugate of N 6 -[4-(carboxymethyl) phenyl]adenosine and compound XII of Figure 10B, wherein R' is CH 3 a conjugate of N 6 -[4-(carboxymethyl) phenyl)adenosine and compound XII of Figure 10B, wherein R' is (CH2)2SCH 3 a conjugate of N 6 -[4-(carboxymethyl) phenyl]adenosine and compound XII of Figure 10B, wherein R' is CH(CH 3 2 a conjugate of N 6 -[4-(carboxymethyl) phenyl]adenosine and compound XII of Figure 10B, wherein R' is CH 2 CH(CH 3 2 a conjugate of N 6 -[4-(carboxymethyl) phenyl]adenosine and compound XII of Figure 10B, wherein R' is C(CH 3 )CH 2 CH 3 and a conjugate of N 6 (carboxymethyl) phenyl]adenosine and compound XII of Figure 10B, wherein R' is CH 2 C 6 H 5

49. A method for administering the binary conjugated of claim 46, wherein said conjugate is delivered using an administration means selected from the group consisting of intravenous administration, oral administration and cardiac perfusion. A method as claimed in claim 49, wherein said conjugate is administered to said patient prior to a surgical procedure having potential to cause cardiac ischemic damage. WO 98/50047 PCT/US98/09031

51. A method as claimed in claim 49, wherein said conjugate is administered to said patient during a surgical procedure having potential to cause cardiac ischemic damage.

52. A method as claimed in claim 49, wherein said conjugate is administered to said patient following a surgical procedure having potential to result in cardiac ischemic damage.

53. A method as claimed in claim 49, wherein said patient is in need of said treatment due to an anginal condition selected from the group consisting of chronic stable angina, unstable angina, and post myocardial infarction angina.

54. A method as claimed in claim 49, wherein said patient is in need of said treatment due to acute myocardial infarction. A recombinant cardiac myocyte comprising a nucleic acid encoding an adenosine receptor selected from the group consisting of the A3 receptor, the A3 receptor, or the A2a adenosine receptor.

56. A recombinant myocyte as claimed in claim whrerein the myocyte is a chick embryo ventricular myocyte and the adenosine receptor is a human adenosine receptor.

57. A method for determining whether a test compound exerts a cardioprotective effect, comprising: a) providing a recombinant myocyte expressing an adenosine receptor; b) contacting said cells with said test compound; WO 98/50047 PCT/US98/09031 c) exposing cells to ischemic conditions; and d) assessing the presence of said cardioprotective effect, if any, exerted by said test compound.

58. A method as claimed in claim 57, wherein said cardioprotective effect is assessed by determining the number of myocytes killed.

59. A method as claimed in claim 57, wherein said cardioprotective effect is assessed by determining the amount of creatine kinase released from said recombinant cardiac myocytes.

60. A method as claimed in claim 57, wherein said recombinant myocyte is selected from the group consisting of chick embryo ventricular myocytes or adult rat ventricular myocytes.

61. A method as claimed in claim 57, wherein said adenosine receptor is the human A3 adenosine receptor.

62. A method as claimed in claim 57, wherein said adenosine receptor is the human Al adenosine receptor.

63. A method as claimed in claim 57, wherein said adenosine receptor is the human A2a adenosine receptor.